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      Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

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          Abstract

          We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

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          Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell.

          P. Bastiaens (1999)
          Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.
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            Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

            Sean C. Warren, Anca Margineanu, Dominic Alibhai … (2013)
            Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.
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              How many photons are necessary for fluorescence-lifetime measurements?

              Malte Köllner, Jürgen Wolfrum (1992)
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                Author and article information

                Contributors
                Niko Hildebrandt: Role: Academic Editor
                Igor Medintz: Role: Academic Editor
                Russ Algar: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Sensors (Basel)
                Journal ID (iso-abbrev): Sensors (Basel)
                Journal ID (publisher-id): sensors
                Title: Sensors (Basel, Switzerland)
                Publisher: MDPI
                ISSN (Electronic): 1424-8220
                Publication date (Electronic): 19 August 2016
                Publication date Collection: August 2016
                Volume: 16
                Issue: 8
                Electronic Location Identifier: 1312
                Affiliations
                [1 ]Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK; robin.willows08@ 123456imperial.ac.uk (R.J.W.W.); david.carling@ 123456imperial.ac.uk (D.C.)
                [2 ]Institute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK; a.sardini@ 123456imperial.ac.uk
                [3 ]Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK; paul.french@ 123456imperial.ac.uk (P.M.W.F.); christopher.dunsby@ 123456imperial.ac.uk (C.D.)
                [4 ]Centre for Pathology, Department of Medicine, Imperial College London, London W12 0NN, UK
                [5 ]Whole Animal Physiology and Imaging, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UK
                [6 ]The Kinghorn Cancer Centre, Garvan Institute of Medical Research and St Vincent’s Clinical School, University of New South Wales, Darlinghurst, NSW 2010, Australia; s.warren@ 123456garvan.org.au
                Author notes
                [* ]Correspondence: g.chennell13@ 123456imperial.ac.uk ; Tel.: +44-20-8383-8262
                [†]

                These authors contributed equally to this work.

                Article
                Publisher ID: sensors-16-01312
                DOI: 10.3390/s16081312
                PMC ID: 5017477
                PubMed ID: 27548185
                SO-VID: 508fd189-8a9d-4514-8a75-9bf88c6e2e29
                Copyright © © 2016 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 20 June 2016
                Date accepted : 12 August 2016
                Categories
                Subject: Article

                ScienceOpen disciplines: Biomedical engineering
                Keywords: fret,flim,ampk,spheroid,2-photon,biosensor,tcspc,3d culture
                Data availability:
                ScienceOpen disciplines: Biomedical engineering
                Keywords: fret, flim, ampk, spheroid, 2-photon, biosensor, tcspc, 3d culture

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